Hi Gabriel,
Phospho and cytokine staining are not usually done in the same tube, even if you're looking at basal levels (rather than stimulated).
1. You need to fix cells in order to stop the phosphatase activity that would decrease your phospho signals. However, most ICS protocols and a lot of surface phenotyping protocols are written for staining *live*, unfixed cells.
2. ICS is most often done using more gentle perms like saponin, rather than methanol. Methanol is a harsh perm which usually does not preserve 3D epitopes. Antibodies *can* be raised against linear epitopes, but those aren't the most common ICS ones.
So, even if you're just looking at basal levels, you'd have to hunt for both surface and cytokine antibodies that can withstand both fixation and methanol.
If you're stimulating cryopreserved PBMCs, I don't think you can really do it because of stimulation timelines: phospho stims are usually for 15min or so, whereas ICS stims are for 4-6 hr (or overnight), depending on the stim. Phosphos are usually transcription factors that get phosphorylated to direct a cell to make a particular protein/cytokine. So, signalling happens quickly and then turns off quickly, whereas it can take a few hours (even with brefeldin) to build up enough cytokine levels to detect. See Frei et al, (
http://dx.doi.org/10.1038/nmeth.3742 ) Fig 1D for an example of the timing difference between mRNA synthesis and protein levels (and phospho signalling technically would happen upstream of the mRNA synthesis).
Mike